1. Field of the Invention
This invention relates to a process for producing and secreting polypeptides in bacteria by overproduction and secretion of a DsbA or DsbC protein.
2. Description of Related Disclosures
Much research has been conducted in the field of protein folding since the seminal publication of Anfinsen et al., Proc. Natl. Acad. Sci. USA, 47: 1309-1314 (1961) showing that, in vitro, reduced and denatured ribonuclease could refold into the active enzyme with the formation of suitable disulfide bonds. Later, a catalyst responsible for oxidative folding in eukaryotes was discovered, called protein disulfide isomerase (PDI).
Two types of proteins that assist in protein folding have been described: non-catalytic molecular chaperones that presumably prevent improper interactions leading to aggregation and events other than proper folding, and catalysts for two steps in protein folding, cis-trans prolyl isomerization and disulfide bond formation. While clear evidence for an in vivo requirement of prolyl isomerase activity is still lacking, the relatively recent isolation of mutants that are severely defective in disulfide bond formation has confirmed that this latter folding step is catalyzed in vivo.
PDI has been implicated in the catalysis of disulfide bond formation and rearrangement through in vitro dam. Creighton et al., J. Mol. Biol., 142: 43-62 (1980); Freedman et al., Biochem. Soc. Symp., 55: 167-192 (1989). See also Bardwell and Beckwith, Cell, 74: 769-771 (1993). In addition, yeast mutants in PDI that fail to form disulfide bonds in carboxypeptidase Y have been identified, indicating the importance of PDI in disulfide bond formation in vivo. LaMantia and Lennarz, Cell, 74: 899-908 (1993). In prokaryotes, mutations in a specific gene identified in the periplasm of E. coli by three independent investigating groups show a dramatic and pleiotropic decrease in the rate of disulfide bond formation in secreted proteins. The protein encoded by this gene was variously designated as DsbA (disulfide bond) by Bardwell et al., Cell, 67: 581-589 (1991) and Missiakas et al., Proc. Natl. Acad. Sci. USA, 90: 7084-7088 (1993), and as PpfA (periplasmic phosphatase/protein formation) by Kamitani et al., EMBO J., 11: 57-62 (1992). It is hereinafter referred to by the Dsb designation. The secreted proteins affected by this decrease in disulfide bond formation range from proteins endogenous to E. coli such as alkaline phosphatase and OmpA to the recombinant mammalian proteins, urokinase and tissue plasminogen activator. It is now clear that the formation of disulfide bonds is a catalyzed process in both eukaryotes and prokaryotes. For a review on PDI and DsbA, see Bardwell and Beckwith, Cell, supra (1993).
The DsbA protein has a highly reactive disulfide bond, similar to disulfide isomerase. Noiva and Lennarz, J. Biol. Chem., 267: 3553-3556 (1992); Bulleld, Adv. Prot., 44: 125-150 (1993). Its crystal structure has recently been solved (Martin et al., Nature, 365: 464-468 [1993]) and its redox potential has been determined. Wunderlich and Glockshuber, Prot. Sci., 2: 717-726 (1993). It is a significantly stronger oxidant than the cytoplasmic thioredoxin and it more closely resembles the eukaryotic disulfide isomerases. It was found that unfolding DsbA stabilizes the reactive disulfide bond by about 18.9 kJmol.sup.-1. Zapun et al., Biochemistry, 32: 5083-5092 (1993). This result is interpreted to indicate that the disulfide bond destabilizes the folded form of DsbA, thereby conferring its high energy content.
The gene of a second protein involved in disulfide bond formation, called dsbB, was cloned by two independent groups. Bardwell et al., Proc. Natl. Atari. Sci. USA, 90: 1038-1042 (1993) and Missiakas et al., 1993, supra. DsbB is an integral membrane protein spanning the inner membrane. It appears to be involved in the re-oxidation of DsbA and thus may form pan of a chain that links an electron transfer step to the formation of disulfide bonds in the periplasm. Bardwell et al., supra (1993). A third gene for disulfide bond formation, dsbC, was recently identified. Missiakas et al., EMBO J., 13: 2013-2020 (1994); Shevchik et al., EMBO J., 13: 2007-2012 (1994). It encodes a 26-kDa periplasmic protein that can functionally substitute for DsbA.
It has been shown that DsbA is required for the rapid formation of disulfide bonds of periplasmic E. coli proteins in vivo and in vitro. The same was found for the production of recombinant eukaryotic proteins in the periplasm of E. coli, e.g., different serine proteases (Bardwell et al., supra [1993]), antibody fragments (Knappik et al., Bio/Technology, 11: 77-83 [1993]), and fragments of a T-cell receptor. The fraction of the recombinant molecules that becomes correctly folded can be smaller than in the case of natural periplasmic proteins, and is very sequence dependent. Overproduction of DsbA did not help to increase the proportion of correctly folded periplasmic antibody fragments (Knappik et al., supra), indicating that other steps limit their folding in the periplasm. Furthermore, folding of the .alpha.-amylase/trypsin inhibitor from Ragi in the periplasm was not improved when the dsbA gene was co-expressed and DsbA protein co-secreted without reduced glutathione present in the growth medium, but an increase in correctly folded secreted inhibitor was observed by co-expression of dsbA in conjunction with the addition of reduced glutathione to the medium. Wunderlich and Glockshuber, J. Biol. Chem., 268: 24547-24550 (1993). Further, Wunderlich and Glockshuber show no increase in total accumulation of the secreted .alpha.-amylase/trypsin inhibitor protein. Wulfing and Pluckthun, Molecular Microbiology, 12: 685-692 (1994) produced functional fragments of a T-cell receptor (TCR) in the periplasm of E. coli by modest overproduction of DsbA and the E. coli heat-shock proteins at low temperature. The latter was achieved by over-expression of rpoH, which codes for the heat-shock sigma factor, sigma.sup.32. This increased the folding yield of the TCR fragments in the periplasm by about two orders of magnitude. It is not known what the yield is in the absence of rpoH overproduction. U.S. Pat. Nos. 5,270,181 and 5,292,646 disclose recombinant production of heterologous proteins by expression as a fusion protein with a thioredoxin-like protein (such as the thioredoxin-like domain of PDI) for high stability and solubility. JP 60-38771 published Feb. 15, 1994 discloses the expression of a human PDI gene linked to human serum albumin pre-pro sequence and co-expression of this linked gene and a foreign gene encoding a polypeptide. WO 93/25676 published Dec. 23, 1993 discloses the production of disulfide-bonded recombinant proteins using a PDI, preferably a yeast PDI. EP 293,793 published Dec. 7, 1988 discloses a polypeptide with PDI activity ensuring natural disulfide bridge arrangement in recombinant proteins. WO 94/08012 published Apr. 14, 1994 discloses increasing secretion of over-expressed gene products by co-expression of a chaperone protein such as a heat-shock protein or PDI. EP 509,841 published Oct. 21, 1992 discloses increased secretion of human serum albumin from yeast cells using a co-expression system involving PDI and a protein.
There is a continuing need for increasing the total yield of proteins secreted by prokaryotes.
Therefore, it is an object of this invention to provide a method for increasing polypeptide yield using an economically viable method involving physiologically regulating the intracellular environment for enhanced accumulation of foreign polypeptides in bacteria.
This object and other objects will become apparent to those skilled in the art.